This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycosyl composition analysis was performed by combined gas chromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. Methyl glycosides were first prepared from dry sample provided by the client by methanolysis in 1 M HCl in methanol at 80[unreadable]C (18-22 hours), followed by re-N-acetylation with pyridine and acetic anhydride in methanol (for detection of amino sugars). The samples were then per-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80[unreadable]C (0.5 hours). [These procedures were carried out as previously described in Merkle and Poppe (1994) Methods Enzymol. 230: 1-15;York, et al. (1985) Methods Enzymol. 118:3-40.] GC/MS analysis of the TMS methyl glycosides was performed on an HP 6890 GC interfaced to a 5975b MSD, using a All Tech EC-1 fused silica capillary column (30m [unreadable] 0.25 mm ID). For glycosyl linkage analysis, the sample was permethylated, depolymerized, reduced, and acetylated;and the resultant partially methylated alditol acetates (PMAAs) analyzed by gas chromatography-mass spectrometry (GC-MS) as described by York et al (1985) Methods Enzymol. 118:3-40. Initially, an aliquot of the sample after dialysis was suspended in about 200 ul of dimethyl sulfoxide. The samples were then permethylated by the method of Ciukanu and Kerek (1984) Carbohydr. Res. 131:209-217 (treatment with sodium hydroxide and methyl iodide in dry DMSO). The sample was subjected to the NaOH base for 10 minutes then methyl iodide was added and left for 20 minutes. The base was then added for 10 minutes and finally more methyl iodided was added for 20 minutes. This addition of more methyl iodide and NaOH base was to insure complete methylation of the polymer. Following sample workup, the permethylated material was reduced by superdeuteride to reduce methyl ester of uronic acid, and then hydrolyzed using 2 M trifluoroacetic acid (2 h in sealed tube at 121[unreadable]C), reduced with NaBD4, and acetylated using acetic anhydride/trifluoroacetic acid. The resulting PMAAs were analyzed on a Hewlett Packard 5890 GC interfaced to a 5970 MSD (mass selective detector, electron impact ionization mode);separation was performed on a 30 m Supelco 2330 bonded phase fused silica capillary column. MALDI MS The sample was dissolved in deionized water (1mg/ml) and 1 ul of the solution was spotted on a spot of DHB dried from acetonitrile/water (1:1), and subjected to MALDI MS on a Bruker MicroFlex Mass Spectrometer which was run in the positive mode. All masses were calibrated by malto-oligosaccharide controls run immediately before the samples.